Several gram negative pathogenic bacteria have evolved a specialized protein secretion system termed type III or contact dependent that is capable of directing the translocation of bacterial effector proteins into host cells. Salmonella typhimurium encodes a type III secretion system that is essential for these bacteria to gain access to non-phagocytic cells. This system directs the translocation of several bacterial effector proteins such as an exchange factor for Rho GTPases, a tyrosine phosphatase, an actin-binding protein and an inositol phosphate phosphatase. Recent studies have established that components of the Salmonella typhimurium type III secretion system are organized in a supramolecular structure that is embedded in the bacterial cell envelope. This structure, termed the needle complex, is architecturally similar to the hook-basal body complex involved in the assembly of bacterial flagella. The needle complex is likely to be a membrane channel through which substrate proteins are translocated. In this proposal, experiments are outlined to identify all the structural components of the needle complex and the proteins involved in its assembly. The generation of a protein linkage map of all the S. typhimurium type III components using two-hybrid system analysis is also proposed. The information obtained from these experiments will give critical information about the mechanism of type III secretion. It will also give valuable insight on a bacterial virulence strategy that could serve as a target for future antibiotic treatment.